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published_or_final_version / Ecology and Biodiversity / Doctoral / Doctor of Philosophy

South Africa is one of the countries with the largest mining operations in the world. Most of these mines make use of natural dams and rivers in their water supply, which often are being pumped back into the natural environment. The reticulated water pumped back into the system may contain high concentrations of dissolved chemicals, which may lead to the reduction of the endemic organisms. Many of the heavy metals mined in South Africa are highly toxic at very low concentrations, and it is therefore very important to do frequent analysis on the aquatic environment. The value of chemical analysis per se has become limiting, as chemical analysis supplies information on the levels of chemicals at a certain time, and the new trend is to incorporate biological monitoring into existing monitoring strategies. Heat shock proteins are classified as stress proteins and are primarily expressed under stressful conditions, therefore having the potential to be used as possible biomarkers. Cortisol, also a known stress hormone, has been suspected of suppressing the expression of heat shock proteins by replacing the heat shock protein on the glucocorticoid receptor. This leads to reduced levels of heat shock proteins in the organism through a negative feedback mechanism. However, before information on heat shock proteins and cortisol can be successfully incorporated into ecological risk assessment, an understanding of how cortisol influences heat shock protein levels after heavy metal exposure is needed. This study aims to determine what effect cortisol had on the production of a specific member of the Hsp70 class of heat shock proteins at different time intervals after exposure of Oreochromis mossambicus to various metals. Proteins were determination using the Bradford method, while protein separation was done using Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis. Thereafter, separated proteins were subjected to Western blotting and immunoblotting in order to quantify the different Hsp70 family members in the hepatic tissue. Cortisol levels were determined using a commercially available Cortisol ELISA Test Kit. Hsp70 accumulation occurred in only two exposure groups, namely the cadmium and chromium exposure groups. Accumulation of Hsp70 demonstrated an increase in accumulation rates at the 24 hour time interval of the 10% cadmium exposure group, with accumulation remaining relatively constant in the 20% cadmium exposure group. Accumulation of Hsp70 occurred only at the 48 and 96 hour time intervals in the 10% chromium exposure group. The accumulation of Hsp76 and Hsp74 were observed to follow the same pattern throughout the 96 hour exposure. In the 10% exposure groups accumulation of both Hsp76 and Hsp74 indicated an increase in accumulation rates at the 72 hour of groups exposed to chromium and nickel, whereas the accumulation of Hsp76 and Hsp74 remains constant after exposure to cadmium. In the 20% cadmium exposure group, an increase in Hsp76 and Hsp74 accumulation was observed at the 24 hour time interval, whereby accumulation of Hsp76 and Hsp74 remains constant in both the chromium and nickel 20% exposure groups. Males accumulated higher levels of Hsp70 members than females in the cadmium and chromium exposure groups. In the 10% exposure groups the females accumulated higher levels of Hsp74, whereas the males accumulated higher levels of Hsp74 in the 20% exposure groups. Accumulation of Hsp76 proved to be higher in the females in all exposure groups, compared to the males. Cortisol concentrations remained constant throughout the 96 hour exposure period, with higher cortisol levels observed in the chromium exposure groups. Cortisol concentrations proved to increase at higher concentrations of metal exposure. Cortisol proved to have no significant effect on Hsp70 family member accumulation, except in the cadmium exposure group, where a negative regression was observed. Accumulation of the HSp70 member can be linked to possible metal specificity, due to the fact that Hsp70 accumulated in only two metal species (cadmium and chromium). However, the accumulation of Hsp76 and Hsp74 may possibly prove that the higher concentrations of specific metals leads to early accumulation of heat shock proteins. The higher accumulation levels of Hsp70 in males, compared to accumulation levels in females may be due to the greater need to discard damadged or denatured proteins, whereas higher Hsp74 and Hp76 accumulation levels in females, may be due to the higher levels of reproductive proteins present in females, compared to males. This study therefore concluded that cortisol may have no significant effect on the accumulation of the Hsp70 family members in the liver of fish. / Prof. J.H.J. Van Vuren

M.Sc. / South Africa is renowned for its exploitable mineral resources and continues to be a major player in the world’s mineral markets. The country is well known for containing the world’s largest gold and platinum repositories and electroplating industries, which is the major cause for delivering by-products such as cadmium (Cd), chromium (Cr) and nickel (Ni). Environmental pollution caused by active mining and seepage from closed mines, continuously threatens South African water resources. Such pollution can cause a shift in water chemistry and increase the availability of certain metals to the living organisms of such a system. Even at low concentrations metals are amongst the most toxic environmental pollutants. As a result of their persistence and capacity to accumulate in the environment, metals have a lasting detrimental effect on the ecosystem. Although there is progress in the treatment of metallic wastes, the discharge thereof by industries is still a serious water pollution problem. In the past, chemical analysis of water has proven to be of great use for the detection of pollutants within the environment. The value of chemical analysis alone has become limiting, as chemical analysis supplies information on the levels of chemicals at a certain time. Furthermore, the monitoring of water quality variables often does not reflect long-term events that may play a critical role in determining the ecosystem health. It is now generally understood that measurements of only the physical and chemical attributes of water cannot be used as surrogates for assessing the health of an aquatic ecosystem. The new trend is to incorporate biological monitoring into Abstract existing monitoring strategies. Fish are entirely dependent on the aquatic environment for their survival, rendering them a good monitor of water pollution. Macroscopic changes in organs are preceded by changes at the tissue, cellular or molecular level. These changes are the net result of adverse biochemical and physiological changes within an organism. Histological analysis is a therefore very sensitive parameter and a valuable technique in determining cellular changes in target organs as a result of exposure to stressors. Fish histology can thus be used as an indicator of exposure to contaminants and assess the degree of pollution. Because of the subjective nature of morphological studies correlations with other quantitative studies are difficult. However, incorporation of quantitative methods is essential to the continued development of histopathology as a biomarker of pollution exposure, and to the interpretation of histological responses. The aim of this study is to qualitatively and quantitatively describe the toxic induced histological changes in the selected organs of Oreochromis mossambicus after acute exposure to Cd, Cr and Ni. Fish were exposed to 10% (n=20) and 20% (n=20) of the LC50 concentration of Cd, Cr and Ni respectively under controlled conditions (23 ± 1°C) for 96 hours in an environmental room with a control group (n=5) for each exposure.

Objective was to assess the toxicity of chromium (Cr) contaminated soil (CS) using the earthworm Lumbricus terrestris. Specific aims were to determine: (1) survival (LC50); .(2) immunotoxicity as indicated by lysozyme activity, coelomocyte counts, secretory (SR) and erythrocyte rosette (ER) formation, and phagocytosis; and (3) compare effects of CS exposure with those of Cr spiked artificial soil (AS) . CS Cr concentration was 8.78 mg/g with 98.2% being Cr^3+ and 1.8% being Cr^6+. Using 14 d AS protocol the LC50 was 6.49% CS: AS mixture. CS concentrations of 0.5, 1.0, 2.5 and 5.0% were sublethal, whereas 6.25, 12.5, 25, 50 and 100% CS were lethal. Sublethal exposure caused no immuno- modulation. Exposure to 50% CS: AS mixture for 5 d caused reduced SR and ER formation. Exposure to AS spiked with 0.27% Cr for 5 d resulted in immunomodulation equivalent to 50% CS: AS mixtures. Results indicated the CS to be acutely toxic.

Chromium hydroxide sludge is the end product of the aluminum phosphate coating process used by a truck manufacturer in the Pacific Northwest. This sludge is listed as an (F019) hazardous waste by the Environmental Protection Agency, and must be landfilled in a hazardous waste landfill site. The purpose of this thesis is to evaluate the characteristics of this sludge, and to then recommend suitable methods for handling and disposal. The chemical analysis of the sludge found total chromium to be 5.4% by weight. Industrial hygiene monitoring found the highest concentrations of 0.002 mg/m3 total chromium and 0.00006 mg/m3 hexavalent chromium at the waste treatment plant. The eight hour permissible exposure limit for total chromium is 0.5 mg/m3 and 0.05 mg/m3 for hexavalent chromium. Given the chemical analysis and industrial hygiene monitoring, a management plan is proposed for this truck manufacturer that discusses possible options for handling the (F019) waste, and provides a cost analysis for each of the options. The options that were investigated were onsite treatment, recycling, no change, and delisting. Onsite treatment was not required since the sludge passed the TCLP extraction test. Recycling costs were determined by contacting four recycling firms. Costs were found to be $600/ton and above, which exceeded the current disposal costs of $466/ton. Delisting the sludge from the hazardous waste listing was determined to be the most feasible from a management, cost, and environmental standpoint. Delisting would reduce the costs of handling the waste, would allow a reduction in landfilling costs, and reduce recycling costs. A literature survey provides background information on industrial uses of chromium, health problems associated with chromium, and discusses the regulations that govern disposal of these chromium sludges. / Graduation date: 1991